Hindawi Publishing Corporation

Journal of Analytical Methods in Chemistry

Volume 2013, Article ID 581093, 8 pages
http://dx.doi.org/10.1155/2013/581093

Review Article
Several Affinity Tags Commonly Used in
Chromatographic Purification

Xinyu Zhao, Guoshun Li, and Shufang Liang

State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, No. 17, Third Section of Renmin South Road,
Chengdu 610041, China

Correspondence should be addressed to Shufang Liang; zizi2006@scu.edu.cn

Received 12 July 2013; Revised 11 November 2013; Accepted 2 December 2013

Academic Editor: Kiran Kumar Doddapaneni

Copyright © 2013 Xinyu Zhao et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Affinity tags have become powerful tools from basic biological research to structural and functional proteomics. They were widely
used to facilitate the purification and detection of proteins of interest, as well as the separation of protein complexes. Here, we mainly
discuss the benefits and drawbacks of several affinity or epitope tags frequently used, including hexahistidine tag, FLAG tag, Strep II
tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding
protein (MBP), S-tag, HA tag, and c-Myc tag. In some cases, a large-size affinity tag, such as GST or MBP, can significantly impact
on the structure and biological activity of the fusion partner protein. So it is usually necessary to excise the tag by protease. The most
commonly used endopeptidases are enterokinase, factor Xa, thrombin, tobacco etch virus, and human rhinovirus 3C protease. The
proteolysis features of these proteases are described in order to provide a general guidance on the proteolytic removal of the affinity
tags.

1. Introduction the application and the requirement for specificity, solubility,

The expression and purification of recombinant proteins have
become increasingly common for characterizing structure
and function of proteins in recent years. There is a need to
purify the protein of interest to obtain enough concentration
with high purity before its function, structure and interac-
tions with other proteins can be studied. Various methods
have been used to enrich proteins of interest from crude bio-
logical extracts. The most effective method is affinity purifica-
tion, whereby the protein of interest is enriched by virtue of its
specific binding properties to an immobilized ligand function
in a fashion similar to that of antibody-antigen interactions.
Affinity or epitope tags are peptide sequences, which are
extremely powerful tools and often appended to the target
protein of interest. Initially affinity tags have been devised to
purify recombinant proteins, but now they are also used in
western blot, immunohistochemistry (IHC), immunoprecip-
itation (IP), flow cytometry (FCM), protein localization, and
so forth. However, each tag has its own distinct advantages
and disadvantages [1, 2], which are important to consider
before the final selection of tag to be used. This depends on
binding, and elution conditions.
Generally, tags used to improve the production of recom-
binant proteins can be roughly divided into purification and
solubility tags [3]. Affinity tags include enzymes, protein
domains, or small polypeptides and most of which bind with
high specificity to a range of substrates, such as carbohy-
drates, small biomolecules, metal chelates, antibodies, and
so forth, to allow rapid and efficient purification of proteins.
While the solubility tags enhance the proper folding and solu-
bility of a protein, they are frequently used in tandem with an
affinity tag to aid purification. In this context, we summarize
the features and applications of several common affinity tags
which are available for prokaryotic and eukaryotic protein
expression systems (Table 1).
2. Affinity Tags and Their Features
Expression of recombinant protein in Escherichia coli (E. coli)
or mammalian cells as a fusion protein with neighboring
affinity tag is one of the most popular methods for purifica-
tion of protein or protein complex. Affinity tags are artificial
2 Journal of Analytical Methods in Chemistry

Table 1: Common widely used affinity tags for purification of recombinant proteins.

Affinity tag Length (aa) Size (kDa) Matrix

Hexahistidine (6x His) 6 (generally) 0.84 Metal ions (Ni , Co2+ , Cu2+ , Zn2+ , Fe3+ )
2+

Glutathione S-transferase (GST) 211 26 Glutathione

FLAG 8 1.01 Anti-FLAG mAb
Streptavidin-binding peptide (SBP) 38 4.3 Streptavidin
Strep II 8 1.06 Strep-Tactin (modified streptavidin)
Maltose-binding protein (MBP) 396 42 Amylose
Calmodulin-binding peptide (CBP) 26 2.96 Calmodulin
Chitin-binding domain (CBD) 51 5.59 Chitin
S 15 1.75 S-protein of RNase A
HA 9 1.1 Anti-HA epitope mAb
c-Myc 11 1.2 Anti-Myc epitope mAb

NH2 Affinity tag Spacer Target protein COOH

(a)
Biological extract

Binding
N-terminal tagged
fusion protein

Nonspecific binding COOH

contaminant

Affinity Affinity ligand COOH

matrix
Tag
COOH
Target protein
Washing and Endopeptidase
eluating

Specific binding recombinant proteins (including interaction partners)

and nonspecific binding contaminants

Other affinity steps (e.g., removal of tag with endopeptidase) or analyses

(e.g., identification of interaction proteins with mass spectrometry)

Interaction partner
Unbound fraction
(b)

Figure 1: (a) Schematic illustration of the N-terminal tagged fusion protein. The spacer represents an endopeptidase cleavage sequence and/or
solubility and folding enhancers. (b) Principle of fusion protein affinity purification and removal of the tag (only for N-terminal tagging). The
interaction proteins will be copurified with the tagged fusion protein under native conditions.

polypeptides which were usually grafted either onto the N- or
C-terminus of a target protein through inserting the cDNA
sequence which encoded the tag peptide into a matching
open reading frame of the target protein (Figure 1). In addi-
tion to facilitating the purification of recombinant proteins,
affinity tags can also enhance the yield, solubility, and even
folding of the target partners [2, 4]. The small-size tags
(e.g., 6× His, FLAG, Strep II, and CBP) have the benefits
of minimizing the effect on the structure, activity, and char-
acteristics of the recombinant protein, and therefore usually
there is no need to remove. Large-size tags, including MBP
and GST, have positive influences on protein solubility and
Journal of Analytical Methods in Chemistry
expression efficiency, but the immunogenicity and the more
consumption of cell metabolic energy in overexpressing cells
are the major drawbacks compared with the small-size tags.
The hexahistidine tag (6× His-tag) is the most frequently
used affinity tag for protein enrichment. His-tagged proteins
can be purified easily by the chelated metal ions as affinity
ligands. The basis for affinity purification is known as immo-
bilized metal affinity chromatography (IMAC) [5]. His-tag
can bind best to IMAC resin in near-neutral buffer conditions
(physiologic pH and ionic strength), and thus the fusion
proteins can be eluted with binding buffer containing certain
concentrations of imidazole. If possible, the elution is also
accomplished with low pH (e.g., 0.1 M glycine-HCl, pH 2.5)
or an excess of strong chelator (e.g., EDTA). The 6× His-
tag has several merits, including a smaller size, absence of
electric charge, low levels of toxicity, and immunogenicity
[3]. His-tagging provides good yields of fusion protein from
inexpensive, high capacity resins with moderate purity from
E. coli extracts but relatively poor purification from mam-
malian cell extracts [5]. For example, we expressed a recom-
binant protein FAM92A1-289 fused with 6× His-tag in E. coli
and purified it using Ni2+ -charged affinity resin for further
function studies [6]. In the prokaryotic expression system,
most of the recombinant His-tagged protein exists in form
of inclusion body, and finally about 4 mg of FAM92A1-289
protein was obtained with high purity from 1 L of E. coli
culture [6].
The maltose-binding protein (MBP) was one of the affin-
ity tags to be used for the purposes of overcoming problems
associated with the expression and purification of fusion
proteins [7]. Generally, recombinant proteins tagged with
MBP can alleviate toxicity and improve expression level and
protein solubility [8–10]. MBP tagging may produce a higher
percentage of recombinant protein than that the polyhistidine
tag does [11, 12]. However, the disadvantage of MBP is the size
and immunogenicity of the affinity tag, which complicates
any downstream application. The purification of MBP-tagged
proteins is achieved by conventional amylose resin-based
chromatography. The elution of the MBP-fused proteins is at
neutral pH using mild maltose-containing buffer conditions
[13]. MBP tag is effective when placed on the N-terminal
or C-terminal end of target proteins. However, because the
large size of this MBP tag puts a heavy metabolic load on the
host cell, the target protein remains insoluble or is prone to
aggregation when the MBP tag is removed [14]. In addition,
recently a novel SUMO fusion tag appears to enhance protein
expression and solubility in prokaryotes and eukaryotes [14,
15].
The glutathione 𝑆-transferase (GST) tag is another well-
established affinity tag based on the strong affinity of GST
for immobilized glutathione [16]. The GST tag is best suitable
for use in prokaryotic expression because GSTs are a family
of multifunctional cytosolic proteins that are present in
eukaryotic organisms but generally not found in bacteria
[17]. Similar to the MBP tag, GST tag has long been used to
increase the solubility of fusion proteins in E. coli [18]. GST-
tagged proteins are captured by immobilized glutathione and
then are eluted under mild, nondenaturing conditions using
reduced glutathione [19].
3
The Strep-tag is an octapeptide that binds to streptavidin
[20, 21]. Streptavidin was also optimized to increase peptide-
binding capacity, which resulted in the development of Strep-
Tactin. The streptavidin derivative, namely, Strep-Tactin,
leads to a higher affinity for Strep II tag [22–24]. Strep II
tag does not interfere with folding or bioactivity and does
not induce protein aggregation either. Strep fusion proteins
can be captured by Strep-Tactin ligand immobilized on the
base matrix and purified in one step from crude cell extracts
under physiological conditions, and thus the tag is especially
applied to the generation of functional proteins or protein
complexes [25]. At the same time, Strep II tag may provide
an acceptable compromise of excellent purification with pure
yields at a moderate cost [6]. In addition, the 38-amino acid
streptavidin-binding peptide (SBP) tag was developed, which
binds to streptavidin more tightly than the Step-tag II and the
native tag [26]. The Strep-tagged or SBP-fused proteins can
be dissociated from the ligand covalently attached to agarose
resin by elution buffer with biotin or desthiobiotin [27, 28].
The calmodulin-binding peptide (CBP) tag was invented
to purification of recombinant protein from bacteria based on
high affinity for calmodulin with nanomolar affinity at phys-
iological conditions in the presence of calcium [29]. The CBP
tag is derived from the C-terminal fragment of human muscle
myosin light-chain kinase and thus is not recommended for
purification of fusion proteins in eukaryotic cells because
endogenous proteins can interfere with calmodulin in a
calcium-dependent manner [30]. Similar to the hexahistidine
and the Strep II tags, CBP tag has a negligible impact on
the biological activity or physical characteristics of the target
partner. CBP-fused proteins are eluted with a strip of calcium
from the environment under very moderate buffer conditions
(e.g., 2 mM EGTA, pH 8.0) [31].
The chitin-binding domain (CBD) from Bacillus circu-
lans consists of 51 amino acids, which is commonly used
as tags for affinity purification of recombinant proteins in
combination with self-splicing inteins in bacterial systems
[32]. Following affinity selection of the fusion protein on a
chitin matrix, the intein undergoes specific cleavage by a thiol
reagent or pH and temperature shift which releases the target
protein from the chitin-bound tag [3].
The FLAG tag is a hydrophilic octapeptide epitope tag
that was introduced to purify fusion proteins [33]. It is likely
to be located on the surface of a fusion protein because
of its hydrophilic nature and therefore is more likely to be
accessible to antibodies. FLAG tag binds to several specific
anti-FLAG monoclonal antibodies such as M1, M2, and M5
with different recognition and binding characteristics [34,
35]. FLAG fusion proteins can be recognized by monoclonal
antibody with calcium-dependent (e.g., M2) or calcium-
independent manner [32]. In particular, the tag appended
to the N-terminus of the fusion protein is necessary for the
immunoaffinity purification with M1 monoclonal antibody,
while M2 is position-insensitive. The elution of the FLAG-
tagged proteins is performed with FLAG peptide (e.g., 3×
FLAG peptide) or low pH glycine buffer (e.g., 0.1 M glycine,
pH3.5) [36].
The S-tag system is based on the specific binding between
15-amino acids S-tag and S-protein, both of which are derived
4 Journal of Analytical Methods in Chemistry
from pancreatic ribonuclease A (RNase A). Any protein fused
with the S-tag can be conveniently purified, detected, and
even quantified [37–39]. However, the elution of S-tagged
proteins is performed under highly stringent condition in the
presence of 3 M NaSCN, 3 M MgCl2 , or 0.2 M citrate (pH 2).
Besides the affinity tags described above, other polypep-
tides such as the HA tag and the c-Myc tag are well charac-
terized and highly immunoreactive tags which are generally
used for the separation of tagged proteins from cell culture
supernatants and cell lysate under neutral pH conditions
and thus are handy tools for coimmunoprecipitation (co-IP)
but are also easily detected via western blot. Moreover, they
are small and thus unlikely to interfere with the bioactivity
and function of the fusion partner proteins. HA tag comes
from human influenza hemagglutinin (HA) corresponding
to amino acids 98–106 and is a strong immunoreactive epi-
tope making it popular to isolate, purify, detect, and track
the protein of interest [40, 41]. The recombinant HA-tagged
proteins can be separated by highly specific anti-HA mono-
clonal antibody that is covalently immobilized on resin. The
HA-tagged proteins can be eluted by mild elution approach
with HA epitope at 1 mg/mL in TBS. On the other hand, three
chemical elution options are available: 0.1 M glycine (pH 2–
2.8), 3 M NaSCN, or 50 mM NaOH.
The c-Myc tag originates from the c-myc gene product.
The recombinant protein tagged with c-Myc tag can be recog-
nized by a well-known high-affinity 9E10 antibody [42].
Though it can be added to the C-terminus or N-terminus
of a protein, it is not recommended to append the c-Myc
tag directly behind the signal peptide of a secretory protein
because the tag can interfere with translocation into the
secretory pathway. In any case, the c-Myc tag can be used in
many different assays such as subcellular localization studies
by immunofluorescence or detection by western blot. Under
native conditions, the elution of c-Myc-tagged proteins can be
achieved by the addition of the c-Myc tag peptide (0.5 mg/mL
in PBS) which competes with the recombinant proteins.
3. Combinatorial Tagging Strategy and
the Studies of Protein Interacting Partners
Protein complexes and protein-protein interactions consti-
tute the functional bases of the life activities within the
cell. Many tag combinations have been developed since the
tandem affinity purification (TAP) technique appeared at late
1990s [43]. TAP-tagging, which employs two sequential affin-
ity purification steps, can significantly reduce the chance of
contaminants retained in the eluate. Double-affinity tag is
an efficient approach for the purification protein complexes
under native conditions [44, 45]. As a powerful tool to sep-
arate interacting protein complex, TAP-tagging strategy is
widely used in the studies of protein interaction networks.
The combination of TAP technique with mass spectrometry
(MS) has been widely adopted as a highly efficient method
to identify and characterize the components of protein com-
plexes [46–49]. For example, we developed a TAP tag system
containing a FLAG and a CBP tag to purify binding partners
with a bait protein 14-3-3𝜀 in mammalian cells, and a new
interacting protein HSP70 was identified by two steps of
affinity purification [48].
Various combinations of different tags have been reported
so far, such as His and FLAG, His and Strep II, FLAG and
Strep II, and so on [50, 51]. Fortunately, many TAP expression
vectors are commercially available today. Altogether, the
adoption of tag in TAP strategy needs to carefully determine
according to the advantages and disadvantages of various
tags and the characteristics of a target protein. To choose an
effective combination, it is normally necessary to consider the
abilities of the tags to improve the yield, enhance the solu-
bility, and facilitate the purification of their fusion partners.
Additionally, if affinity tags have the potential to interfere
with structural or functional studies, the fused tag must be
removed from the bait protein as follows.
4. Removal of Affinity Tags
The use of affinity tag for the purification of proteins in both
prokaryotic and eukaryotic expression systems is a well-
accepted method. In theory, it cannot be excluded that affinity
tags, especially those with large size, may have the potential
to interfere with the structure and function of the proteins.
If this circumstance happens, measures should be made for
removing them. Any affinity tag, whether small or large,
can be easily removed by introducing a specific protease
recognition sequence between the tag and target protein
(Figure 1(b)). The most frequently used endopeptidases are
enterokinase, factor Xa, thrombin, tobacco etch virus (TEV),
and human rhinovirus 3C protease. The advantages and dis-
advantages of these endopeptidases were thoroughly dis-
cussed in the previous works of the literature [1, 52–54].
Moreover, other endopeptidases (e.g., PreScission and Sor-
tase A) and exopeptidases (e.g., DAPase, Aeromonas ami-
nopeptidase, aminopeptidase M, and carboxypeptidase A
and B) were described exhaustively for the removal of affinity
tags from recombinant proteins [1, 52].
The small-size tags such as 6× His, FLAG, Strep II, and
CBP usually do not need to be removed for downstream
applications following purification. Enterokinase possesses
trypsin-like activity and specifically cleaves after lysine res-
idue at a canonical recognition sequence (DDDDK↓) [55].
Enterokinase may sometimes cleave at other basic residues,
depending on the conformation of the protein substrate
[56–60]. In addition, the cleavage efficiency of enterokinase
is closely associated with the amino acid residue after down-
stream of the recognition site [61, 62]. It will not cleave at
the recognition site if the recognition sequence is followed
by proline. However, the addition of urea (1–4 M) can greatly
improve enterokinase cleavage specificity and reduce adven-
titious cleavage [63]. In particular, FLAG tag (DYKDDDDK)
contains the enterokinase cleavage site (underlined) which
allows the removal of affinity tag after purification.
As the enterokinase, both thrombin and factor Xa are
trypsin-like serine proteases which will cleave peptide bonds
on the carboxyl side of a basic amino acid residue. Factor
Xa cleaves after the arginine residue in its preferred cleavage
site (I-E/D-G-R↓) and occasionally cleaves at other sites [64–
66]. Similar to the enterokinase, factor Xa will not cleave at a
Journal of Analytical Methods in Chemistry
site followed by proline or arginine. For the most common
recognition sequence (LVPR↓G) or (LVPR↓GS), thrombin
will selectively cleave after arginine residue [54]. Thus, unlike
enterokinase and factor Xa, thrombin cleavage may result
in the retention of one or two amino-terminal amino acid
residues in protein of interest.
The TEV protease is a highly site-specific cysteine endo-
protease. Its optimum recognition sequence is E-N-L-Y-F-
Q↓-G/S and the cleavage occurs between the glutamine and
glycine/serine residues [67].
Sortase A is a prokaryotic thiol transpeptidase that can
hydrolyze the fusion proteins by recognizing a carboxyl-
terminal sorting signal sequence (LPET↓G) and cleaving the
threonine-glycine peptide bond [68, 69]. The cleavage activity
of the enzyme can be stimulated by calcium ions [70], and an
additional affinity step is needed for on column tag removal
such as using immobilized sortase A [71].
PreScission protease is a GST tagged human rhinovirus
(HRV) type 14 3C protease and specifically recognizes the
amino acid sequence LEVLFQ↓GP or its subset of sequences
which include the core amino acid sequence (underlined),
cleaving between the glutamine and glycine residues [72, 73].
The recombinant enzyme is specifically designed to facilitate
removal of the protease by allowing simultaneous protease
immobilization and cleavage of GST affinity tag.
Besides the tag cleavage methods described above,
removal of the tag without using a protease is also feasible by
introducing a protein element with self-splicing capacity into
a variety of tag-based purification systems. These elements are
naturally self-splicing proteins called inteins that can excise
themselves from the parent protein [74, 75]. Inteins can be
designed at N- or C-terminal splice junctions to obtain self-
cleaving inteins, which can then be used to achieve self-
cleaving of various affinity tags. The specific applications have
been comprehensively reviewed by other authors [76–78].
5. Conclusions
Affinity purification is an approach of isolating biomoleculars
from cell extracts based on a highly specific interaction as
that between antigens and antibodies, as well as receptors and
ligands. Affinity tag is absolutely necessary for many appli-
cations in life sciences, including the purification of protein
of interest. Although each tag has its specific advantages and
disadvantages in purification efficiency, the versatile affinity
purification systems with different affinity tags are powerful
to isolate recombinant protein and protein complexes. To
sum up, there is no universal affinity purification system for
exploring different bait protein and its binding partners at
present. So far, it is feasible to generate multiple purified
human proteins or protein domains for affinity proteomics
and large-scale structural genomics studies by now [79].
Acknowledgments
This work was financially supported by the National
Key Basic Research Program of China (2011CB910703,
2013CB911303), the National 863 High Tech Foundation
5
(SS2014AA020608), and the Natural Science Foundation of
China (30800581, 31071235). This research was also funded
by the New Century Excellent Talents in University (NCET-
10-0595) and the Specialized Research Fund for the Doctoral
Program of Higher Education (20120181110025).
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